The goals of our project are to isolate prostate epithelial populations, including prostate stem cells, and to evaluate the tumorigenic and the metastatic capacity of such populations upon transformation by deletion of the tumor suppressor, PTEN, either alone or in combination with alteration of the p53 tumor suppressor. PTEN deletion or inactivation is common in human prostate cancer, and PTEN deletion in mouse prostate epithelium has been shown to mimic human prostate cancer in the production of adenocarcinoma with a luminal phenotype, which is capable of giving rise to androgen independent secondary tumors. The addition of p53 deletion has been shown to result in more highly penetrant and rapidly evolving disease as a result of decreased cellular senescence. Because PTEN deletion is an embryonic lethal event, loss of PTEN has been engineered to be specific for prostate epithelium, using CRE-dependent excision. We have established appropriate strains of mice carrying genetically modified loci and validated methods to obtain specific PTEN and/or p53 excision both in vivo and in vitro. Another common genetic mutation observed in prostate cancer is the rearrangement of the ERG transcription factor adjacent to the androgen-regulated TMPRSS2 promoter, which leads to abnormal expression of ERG. We are currently developing a transgenic strain of mice carrying a BAC plasmid, which contains a recombineered human TMPRSS2-ERG translocation. These two models, one inducible and the other constitutive, either alone or in combination, provide the source of tumor initiating cells in this study. Existing mouse models of prostate cancer demonstrate relatively little metastasis and usually to lymph nodes only. Possible explanations for this observation are, 1) polyclonal tumor formation in transgenic models leads to mortality due to tumor burden before the conditions for metastatic spread occur, or 2) the wrong precursor population is transformed, or 3) transforming events are missing that lead to the metastatic phenotype. An approach that allows the above possibilities to be systematically investigated is based upon orthotopic transplantation of selected tumor initiating cells. We have successfully transplanted prostate epithelial cells into the dorsal prostate of the mouse and longitudinally followed the progression of the transplanted cells using bioluminescent imaging. Orthotopic transplantation will be used to evaluate tumorigenic and metastatic potential in various prostate epithelial populations, separated on the basis of cell surface markers and transformed with defined genetic events.